1. Field of the Invention
The present invention relates generally to the fields of medical glues and adhesives. More particularly, it concerns methods and compositions for sealing of wounds and incisions. In certain aspects, the invention concerns adhesion of two or more tissue samples using proteinaceous, and/or lipoproteinaceous compositions conjugated and/or mixed with a photosensitizer or dye upon irradiation.
2. Description of Related Art
Healing and sealing tissue wounds remains a problem in medical practice. To enhance tissue healing, much effort has gone towards producing a biocompatible slow release formulation which can be introduced into a tissue defect and that will release biologically active growth factor at a steady rate for the required time. These formulations were designed to promote tissue growth or healing. Examples of these vehicles include biodegradible gelatin hydrogel (Yamamoto et al., 2000), hyaluronan (Mohammad et al., 2000), fibrin glue (Cheng et al., 1998), fibrin derivatives (Sakiyama-Elbert and Hubbell, 2000), alginate microspheres (Nehra et al., 1999), carbopol gel (Sheardown et al., 1997), derivatized dextrans (Tardieu et al., 1992), calcium alginate beads (Downs et al., 1992). It should be noted that many of these slow release vehicles do not actually bind to the tissue, they merely xe2x80x9csitxe2x80x9d in the defect and slowly biodegrade. However, a TGF-beta 1 and indocyanine green albumin solder have been used in incisions in pig skin (Poppas et al., 1996). A significant increase in wound healing strength was found using TGF-beta containing solder compared to solder alone.
To replace or promote healing in damaged tissue, various forms of tissue transplants have been conducted. However, cell transplantation often requires that that the donor cells retain their polarity and function, avoid formation of clumps or multilayers, and maintain their viability. In certain tissues, a biodegradable matrix has been used to transplant cells. Three-dimensional cell culture systems with various attachment substrates offer new probabilities for long-term viability and donor cell functions (Wintermantel et al., 1992; Fawcett et al., 1995; Spier and Maroudas, 1991; Peshwa et al., 1996; Rezai et al., 1997; Ruoslahti and Hayman, 1992; Spector et al., 1993; Hoffman, 1994; Kleinman et al., 1998). Successful use of 3-dimensional micro-carriers for transplantation to the liver (Wintermantel et al., 1992) and brain (Fawcett et al., 1995) has been reported by several investigators. In 3-dimensional micro-carriers, the cultured cells are distributed at the outer surfaces and within the body of the particles (Spier and Maroudas, 1991). In a 3-dimensional carrier, more cell contacts are generated compared with a monolayer state, thereby facilitating cell proliferation and spreading (Peshwa et al., 1996; Rezai et al., 1997). The chemistry of the extracellular matrix itself can also modulate various aspects of cell behavior, including adhesion, proliferation, and migration (Ruoslahti and Hayman, 1992).
Successful retinal pigment epithelium (RPE) transplantation requires cell attachment to a substrate prevents RPE apoptosis and de-differentiation after transplantation (Tezel and Del Priore, 1997; Ho et al., 1996). Subretinal provision of RPE cells has been carried out in the form of a cell suspension, RPE patches, or RPE cells grown on artificial substrates (Li and turner, 1991; Sheedlo et al., 1989; Gabrielian et al., 1999; Bhatt et al., 1994). Cell suspension provision has the limitations of reflux from the iatrogenic retinotomy site and irregular distribution of the donor cells in the subretinal space (Wongpichedchai et al., 1992). Retinal pigment epithelium patch grafts, although probably the most physiologic, have not been shown to proliferate in vivo (Gouras et al., 1994; Berglin et al., 1997).
Sealing tissue wounds usually involves sutures and other mechanical seals. Alternative methods to the traditional mechanical means of closing incisions, wounds, and anastomoses have received attention. These may be divided into three groups: first, biological glues (Basu et al., 1995) such as fibrin sealant (Sierra, 1993) and gelatin-resorcinol glue (Albes et al., 1993); second, a technique known as laser tissue welding, which relies on carbon dioxide (Rooke et al., 1993) or Nd:YAG (Back et al., 1994) lasers to produce thermal effects to attach tissue surfaces; and third, chromophore-assisted laser welding (Bass and Treat, 1995) using protein solders that contain a light-absorbing dye together with a laser that emits the appropriate wavelength light. This pairing is most commonly that of fluorescein and a 532-nm frequency-doubled Nd:YAG laser, or indocyanine green and an 805-nm diode laser (Wright and Poppas, 1997).
Alternative tissue adhesives have drawbacks. Cyanoacrylate glues, which have been most frequently used in ophthalmology (Leahey et al., 1993) can be toxic, causing inflammatory reactions and are nonbiodegradable (Siegal and Zaidman, 1989). Fibrin sealants (Spontnitz, 1995) are not particularly effective, form bonds of insufficient strength (Basu et al., 1995; Siedentop et al., 1988), present the possibility of viral infection if prepared from pooled human plasma, and may inhibit would healing (van der Ham et al., 1993). Resorcinol gelatin sealants (Albes et al., 1993) can damage tissue because they contain formaldehyde (Ennker et al., 1994). However, laser-activated tissue solders allows safe preparation and sterilization of the material, because it is activated only under laser illumination and is thought unlikely to lead to tissue toxicity (Bass and Treat, 1995).
Laser tissue welding have been used in urology (Kirsch et al., 1997), vascular surgery (Ashton et al., 1991), neurosurgery (Menovsky et al., 1995), and orthopedics (Forman et al., 1995). Ophthalmologic applications of laser welding with chromophore-assisted protein solder have included sealing cataract incisions (Eaton et al., 1991) and scleral tunnel incisions (Kim et al., 1995) and bonding synthetic epikeratoplasty lenticules to the cornea (Gailitis et al., 1990).
Laser tissue welding without added dye must proceed through a purely thermal mechanism (Schober et al., 1986), whereby the edges of the collagen are partially xe2x80x9cunraveledxe2x80x9d and can then recombine to form noncovalent bonds (Pearce and Thomsen, 1993). It was thought that dye-assisted welding with protein solders also proceeded through a thermal mechanism, with the chromophore-absorbing energy, releasing it as heat, denaturing the protein in the solder and forming noncovalent bonds between the added protein solder and the tissue collagen (Small et al., 1997). A mixture of cryoprecipitated fibrinogen and a dye that absorbs laser energy and releases it in the form of heat at the wound interface has been used in tissue adhesion (Moazami, et al., 1990; Oz et al., 1990).
However, results with the two dyes most commonly used for tissue welding, fluorescein and indocyanine green, have produced evidence that photochemical processes occur as well. It has been reported that fluoresceindextran in the rat mesentery lymphatics when illuminated produce changes that could be attributed to singlet oxygen (Zhang et al., 1997). Studies with indocyanine green in vitro have shown that it has a triplet yield of 0.11, and singlet oxygen can be detected by time-resolved luminescence techniques (Baumier et al., 1997; Fickweller et al., 1997). Laser welding with a biologic tissue glue consisting of 18% fibrinogen with 2.6 mg/ml r-5-P showed reduction of the weld strength in the presence of azide which is evidence of singlet oxygen involvement in the weld formation (Khadem et al., 1994).
These chemicals that cause photo-oxidative effects when exposed to visible light have been called xe2x80x9cphotosensitizersxe2x80x9d (Chacon et al., 1988; Tanielian C., 1986; Foote, C. S., 1976). There are two main classes of photosensitizer: tetrapyrroles including porphyrins, chlorins, bacteriochlorins, phthalocyanines, naphthalocyanines, texaphyrins, verdins, purpurins; pheophorbides, etc; and non-tetrapyrrole dyes, including flavins, xanthenes, thiazines, selenium and tellurium analogues of thiazines, azines, triarylmethanes, etc. Fluorescein is a xanthene but is not considered a photosensitizer because it releases absorbed energy primarily in the form of heat and fluorescence. Some of these dyes have been evaluated with proteins as tissue glues with varying success (U.S. Pat. No. 5,552,452).
Chlorine6 (Ce6) has been investigated as a photosensitizer for photodynamic therapy both as the free dye (Kostenich et al., 1994) and conjugated to proteins, (Schmidt-Erfurth et al., 1997), macromolecules (Soukos et al., 1997) and particles (Bachor et al., 1991). Covalent conjugates between Ce6 and monoclonal antibodies (Hamblin et al., 1996) and poly-L-amino acids (Soukos et al., 1997) for the photodynamic therapy of cancer have been described. Ce6 is usually thought to act as a photosensitizer by transferring energy from the triplet state to the ground state of molecular oxygen, producing the exited singlet oxygen molecule, a process known as type II photosensitization (Ochsner, 1997). Singlet oxygen can then react with certain amino acids in proteins, particularly histidine, tryptophan, tyrosine, cysteine, and methionine (Dubbelman et al., 1978). One mechanism that has been elucidated for the formation of intermolecular protein cross-links is the reaction of oxidized histidine with free amino groups of lysines on neighboring proteins (Verweij et al., 1981), but it is recognized that other mechanisms must operate as well. There is another possible photo-oxidation pathway involving electron transfer from the photosensitizer triplet state producing either a radical cation or a radical anion, which is known as type I photosensitization (Zhang and Xu, 1994). These radical ions can then react further with oxygen producing carbon and oxygen centered radicals and superoxide anions (Laustrait, 1986). A mechanism for the radical mediated cross-linking of proteins involves the formation of dityrosine (Gill et al., 1997) by phenolic coupling of tyrosine residues on neighboring chains.
Despite these advances in the understanding of tissue healing, tissue transplantation and tissue welding mechanisms, their exists a need for improved methods to heal, transplant and/or weld tissue. Improved methods to promote tissue healing or aid in successful tissue transplants would provide significant benefits in the art. Tissue welds with improved strength would resist tearing under stress. Additionally, there exists a need for methods and compositions of tissue welding, wound healing and tissue transplantation that are easy to handle during surgery, and possess a reduced toxicity or scaring potential.
The present invention overcomes the deficiencies of the prior art by providing novel compositions and methods for tissue welding. The invention also provides compositions and methods for administering an active agent to a tissue. Such active agents may be living cells. Thus, in certain embodiments, the invention provides a method to transplant tissue. Such transplanted cells may be formed into a desired shape, such as a monolayer.
The invention first provides a method to weld tissue together, comprising the steps of: applying to at least one tissue a composition comprising at least one photosensitizer and at least one proteinaceous compound or at least one lipid; and irradiating the composition with electromagnetic energy; wherein the irradiating promotes adhesion of the tissue to at least a second tissue. In certain embodiments, the photosensitizer is a cationic azine mon-azo dye or derivative thereof. In certain aspects, the cationic azine mono-azo dye is neutral red or Janus Green. In other embodiments, the photosensitizer is a tri-arylmethane dye or derivative thereof. In particular aspects, the tri-arylmethane dye is Malachite Green, Brilliant Green, Crystal Violet, basic fuschin, pararosaniline acetate, methyl green or new fuschin. In certain aspects, the tri-arylmethane dye is a zwitterionic triarylmethane dye, such as patent blue VF. In other embodiments, the photosensitizer is a tetrapyrrole or a derivative thereof. In certain aspects, the tetrapyrrole is a porphyrin, chlorin, bacteriochlorin, phthalocyanine, naphthalocyanine, texaphyrin, verdin, purpurin or pheophorbide. In some aspects, the chlorin is chlorine6. In other aspects, the phthalocyanine is a Zn(II)-phthalocyanine, an aluminum sulfonated and disulfonated phthalocyanine or a phthalocyanine without a metal substituent. In certain aspects, the naphthalocyanine is a sulfonated aluminum naphthalocyanine. In other aspects, the pheophorbide is a pyropheophorbide. In certain embodiments, the photosensitizer is a cationic thiazine dye or derivative thereof. In particular embodiments, the cationic thiazine dye is Azure A, Azure B, Azure C, Brilliant Green, Crystal Violet or Patent Blue VF.
In certain embodiments, the composition further comprises at least a second photosensitizer. In particular aspects, the at least a second photosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye, a tetrapyrrole, a cationic thiazine dye, xanthine, an anthracenedione, an anthrapyrazole, an aminoanthraquinone, a phenoxazine dye, a phenothiazine derivative, a chalcogenapyrylium dye or derivatives thereof.
In certain embodiments, the composition comprises at least one proteinaceous compound. In certain aspects, the proteinaceous compound comprises at least one peptide, polypeptide or protein. In particular aspects, the protein is albumin, fibrinogen or gelatin.
In some embodiments, the composition is a non-covalent mixture. In other embodiments, at least one covalent bond conjugates the photosensitizer to the proteinaceous material or the lipid. In particular aspects, the covalent bond is part of a linking moeity.
In other embodiments, the composition comprises at least a second proteinaceous compound not covalently conjugated to the photosensitizer. In certain aspects, the proteinaceous compound covalently conjugated to the photosensitizer is the same type as the proteinaceous compound not covalently conjugated to the photosensitizer, such as albumin for both proteinaceous compounds.
In certain embodiments, the ratio of total proteinaceous molecules in the composition and the at least one photosensitizer is from about 100:1 to about 1:100. In certain aspects, the ratio of total proteinaceous molecules in the composition and the at least one photosensitizer is from about 10:1 to about 1:10. In other aspects, the ratio of total proteinaceous molecules in the composition and the at least one photosensitizer is from about 3:1 to about 1:1. In a particular aspect, the ratio of total proteinaceous molecules in the composition and the at least one photosensitizer is about 2:1.
In some embodiments, the composition comprises at least one lipid. In certain aspects, the lipid further comprises at least one proteinaceous compound. In other aspects, the proteinaceous compound is a lipoprotein.
In certain embodiments, the composition further comprises at least one therapeutic agent. In particular aspects, the agent is a chemical, a drug, a proteinaceous molecule, a nucleic acid, a lipid, an antibody, an antigen, a hormone, a nutritional substance, a cell or a combination thereof In certain aspects, the hormone is a growth factor, including but not limited to transforming growth factor beta, basic fibroblast growth factor, epidermal growth factor, vascular endothelial growth factor, nerve growth factor, acidic fibroblast growth factor, insulin like growth factor, heparin binding growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, platelet-derived growth factor, leukemia inhibitory factor or combination thereof. In other aspects, the agent is a cell, including but not limited to an embryonic cell.
In other embodiments, the tissue is skin, bone, neuron, axon, cartilage, blood vessel or cornea. In certain aspects, the second tissue is the same tissue type as the at least a first tissue, while in other aspects, the second tissue is a different tissue type as the at least one tissue.
In certain embodiments, the composition is applied at of from about 10 mg the composition per cm2 of the tissue to about 500 mg the composition per cm2 of the tissue. In particular aspects, the composition is applied of from about 20 mg the composition per cm2 of the tissue to about 100 mg the composition per cm2 of the tissue.
In particular embodiments, the composition has a viscosity of about 40 to about 100 poise before the irradiation.
The invention next provides a method to weld tissue together, comprising the steps of: applying to at least one tissue a composition comprising at least one photosensitizer; and irradiating the composition with electromagnetic energy; wherein the photosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye, a chlorine, a tetrapyrrole, a cationic thiazine dye, or derivatives thereof; and wherein the irradiating promotes adhesion of the tissue to at least a second tissue. In certain embodiments, the photosensitizer is neutral red, Janus Green, Malachite Green, Brilliant Green, Crystal Violet, basic fuschin, pararosaniline acetate, methyl green, new fuschin, patent blue VF12, chlorine6, Azure A, Azure B, Azure C, Brilliant Green, Crystal Violet or Patent Blue VF. In particular aspects, the photosensitizer is Janus Green, Malachite Green or chlorine6.
In certain embodiments, the composition further comprises at least a second photosensitizer. In particular aspects, the at least a second photosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye, a tetrapyrrole, a cationic thiazine dye, xanthine, an anthracenedione, an anthrapyrazole, an aminoanthraquinone, a phenoxazine dye, a phenothiazine derivative, a chalcogenapyrylium dye or derivatives thereof.
In other embodiments, the composition comprises at least one proteinaceous compound or lipid.
In certain embodiments, the method of claim 56, wherein the composition is a non-covalent mixture. In some embodiments, the at least one covalent bond conjugates the photosensitizer to the proteinaceous material or the lipid. In particular aspects, the covalent bond is part of a linking moeity.
In some embodiments, the composition comprises at least a second proteinaceous compound not covalently conjugated to the photosensitizer. In certain aspects, the proteinaceous-compound covalently conjugated to the photosensitizer is the same type as the proteinaceous compound not covalently conjugated to the photosensitizer.
In particular embodiments, the composition further comprises at least one therapeutic agent. In some aspects, the agent is a chemical, a drug, a proteinaceous molecule, a nucleic acid, a lipid, an antibody, an antigen, a hormone, a nutritional substance, a cell or a combination thereof.
The invention further provides a method to deliver a therapeutic agent to at least one living cell, comprising the steps of: applying to at least one cell a composition comprising at least one at least one photosensitizer and at least a therapeutic agent; and irradiating the composition with electromagnetic energy; wherein the irradiating promotes adhesion of the composition to the cell, and wherein the agent is thereby contacted with the cell. In certain aspects, the photosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye, a chlorine, a tetrapyrrole, a cationic thiazine dye, or derivatives thereof. In other aspects, the photosensitizer is neutral red, Janus Green, Malachite Green, Brilliant Green, Crystal Violet, basic fuschin, pararosaniline acetate, methyl green, new fuschin, patent blue VF12, chlorine6, Azure A, Azure B, Azure C, Brilliant Green, Crystal Violet or Patent Blue VF. In particular aspects, the photosensitizer is Janus Green, Malachite Green or chlorine6.
In certain embodiments, the composition further comprises at least a second photosensitizer. In some aspects, the at least a second photosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye, a tetrapyrrole, a cationic thiazine dye, xanthine, an anthracenedione, an anthrapyrazole, an aminoanthraquinone, a phenoxazine dye, a phenothiazine derivative, a chalcogenapyrylium dye or derivatives thereof.
In particular embodiments, the composition comprises at least one proteinaceous compound or lipid.
In other embodiments, the composition is a non-covalent mixture. In some embodiments, at least one covalent bond conjugates the photosensitizer to the proteinaceous material or the lipid. In particular aspects, the covalent bond is part of a linking moeity. In certain aspects, the composition comprises at least a second proteinaceous compound not covalently conjugated to the photosensitizer. In other aspects, the proteinaceous compound covalently conjugated to the photosensitizer is the same type as the proteinaceous compound not covalently conjugated to the photosensitizer.
In some embodiments, the agent is a chemical, a drug, a proteinaceous molecule, a nucleic acid, a lipid, an antibody, an antigen, a hormone, a nutritional substance, a cell or a combination thereof. In particular aspects, the hormone is a growth factor. In other aspects, the growth factor is transforming growth facto beta, basic fibroblast growth factor, epidermal growth factor, vascular endothelial growth factor, nerve growth factor, acidic fibroblast growth factor, insulin like growth factor, heparin binding growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, platelet-derived growth factor, leukemia inhibitory factor or combination thereof. In other aspects, the agent is a cell. In specific aspects, the cell is an embryonic cell.
In certain embodiments, a tissue may comprise, but is not limited to, skin, bone, neuron, axon, cartilage, blood vessel, cornea, muscle, facia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood, embryonic or ascite tissue, and all cancers thereof. In certain embodiments, a cell may comprise, but is not limited to, skin, bone, neuron, axon, cartilage, blood vessel, cornea, muscle, facia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood, embryonic or ascite cell, and all cancers thereof.
The invention additionally provides a tissue glue/biomatrix composition, comprising at least one photosensitizer and at least one proteinaceous compound or at least one lipid. In certain embodiments, the photosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye, a chlorine, a tetrapyrrole, a cationic thiazine dye, or derivatives thereof. In other embodiments, the photosensitizer is neutral red, Janus Green, Malachite Green, Brilliant Green, Crystal Violet, basic fuschin, pararosaniline acetate, methyl green, new fuschin, patent blue VF12, chlorine6, Azure A, Azure B, Azure C, Brilliant Green, Crystal Violet or Patent Blue VF. In particular aspects, the photosensitizer is Janus Green, Malachite Green or chlorine6. In other embodiments, the composition further comprises a therapeutic agent. In certain aspects, the agent is a chemical, a drug, a proteinaceous molecule, a nucleic acid, a lipid, an antibody, an antigen, a hormone, a nutritional substance, a cell or a combination thereof.
The invention next provides a tissue glue/biomatrix composition, comprising at least one photosensitizer, wherein the photosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye, a chlorine, a tetrapyrrole, a cationic thiazine dye, or derivatives thereof. In certain embodiments, the composition further comprises at least one therapeutic agent, a proteinaceous compound or lipid.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Following long-standing patent law convention, the word xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean xe2x80x9cone or morexe2x80x9d in this specification, including the claims.